Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A dual-target dual warhead anti-tumor fusion protein, its coding gene and use

A fusion protein and anti-tumor technology, applied in anti-tumor drugs, peptide/protein components, hybrid peptides, etc., can solve the problems of TRAIL limitation, TRAIL sensitivity difference, etc., and achieve the effects of inhibiting proliferation, low drug toxicity, and inhibiting growth Effect

Active Publication Date: 2019-06-21
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
View PDF7 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although early clinical trials are very encouraging, human recombinant TRAIL (Human recombinant TRAIL, hrTRAIL) is only limited to patients with TRAIL-sensitive tumors, and even tumors from the same tissue have different sensitivities to TRAIL
Therefore, the insensitivity of different tumor tissues to TRAIL limits the clinical application of TRAIL.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A dual-target dual warhead anti-tumor fusion protein, its coding gene and use
  • A dual-target dual warhead anti-tumor fusion protein, its coding gene and use
  • A dual-target dual warhead anti-tumor fusion protein, its coding gene and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0138] Construction of fusion protein Ec-LDP-TRAIL recombinant expression plasmid pET30-Ec-LDP-TRAIL

[0139] Recombinant plasmid pET30a(+)-Ec-ldp-Hr (Guo Xiaofang et al., A bispecific enediyne-energized fusion protein containing ligand-based and antibody-based ligopeptides against epidermal growth factor receptor and human epidermal growth factor receptor2shows potent Clinical0 Antitrivitor, , 16.7:2085-2094) and pET30a(+)-TRAIL (Jie Yang, TRAIL combined with LDM and mechanism of action and expression of TRAIL protein. MS thesis. Peking Union Medical College, 2010.) contain Ec-LDP respectively Genes and TRAIL functional genes are preserved by our laboratory. Two restriction sites, NdeI and XhoI, were introduced by overlapping PCR technology, and the primers were synthesized by Invitrogen.

[0140] P1 (SEQ ID NO:8): 5'-TATA CATATG AACTGTGTGGTGGGCTATATTGG-3' (the underline is the Nde I restriction site, followed by the Ec-ldp start sequence)

[0141] P2 (SEQ ID NO:9): 5'-C...

Embodiment 2

[0146] fusion protein Ec-LDP-TRAIL in Escherichia coli BL21 (DE3) star TM inducible expression

[0147] The identified recombinant plasmid pET30-ec-ldp-trail was transformed into Escherichia coli BL21(DE3)star TM (Invitrogen company product), pick the recombinant transformant (this strain is Escherichia coli), which has been preserved in the General Microorganism Center of China Committee for the Collection of Microorganisms on September 17, 2013 (referred to as CGMCC, address : No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), strain preservation number CGMCCNO.8200) was inoculated into 3ml of LB liquid medium containing 50μg / ml kanamycin, shaken at 37°C Incubate overnight. The next day, they were inoculated at a ratio of 5%, cultured with shaking at 37°C until the OD600 was 0.8, added IPTG to a final concentration of 0.2mM, and induced for 8 hours. An appropriate amount of bacterial liquid was taken, and...

Embodiment 3

[0148] fusion protein Ec-LDP-TRAIL affinity chromatography purification and separation preparation

[0149] 3.1 Extraction of inclusion body protein

[0150]The culture of recombinant bacteria induced by IPTG was centrifuged at 10000 g for 10 min, the supernatant was removed as much as possible, and the bacteria were collected. Resuspend the cells with 20ml of 20mM Tris-HCl per 1L of culture volume. Sonication disrupts cells. Centrifuge at 10,000 g at 4°C for 15 min to collect inclusion bodies and cell fragment proteins, and discard the supernatant. Resuspend the pellet with 40 ml of urea-free 1× binding buffer (20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, pH 8.0). Centrifuge at 10,000 g for 15 min at 4°C, discard the supernatant, and collect the precipitate. Repeat the above steps, resuspend the pellet with 50ml of 1× binding buffer (20mM Tris-HCl, 0.5M NaCl, 20mM imidazole, 8M urea, pH8.0), and ice-bath for 1h to completely dissolve the inclusion body protein. Centrifug...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides an anti-tumor fusion protein, which has the following primary amino acid sequence structure: EGFR-specific targeting oligopeptide-connecting peptide-lidamycin apoprotein-connecting peptide-TRAIL functional peptide fragment, wherein the EGFR-specific targeting oligopeptide has an amino acid sequence shown in a SEQ ID NO: 1, the lidamycin apoprotein has an amino acid sequence shown in an SEQ ID NO: 2, and the TRAIL functional peptide fragment has an amino acid sequence shown in an SEQ ID NO: 3. The fusion protein is an EGFR and DR4 / DR5 targeted bispecific fusion protein, and contains an EGFR targeted ligand oligopeptide, and the sensitivity of tumor cells to TRAIL and the natural drug resistance of reverse tumor cells to TRAIL are enhanced by an antagonistic EGFR signaling pathway. The invention also provides a coding gene of the fusion protein, an intensive fusion protein assembled with a lidamycin chromophore, and a pharmaceutical application of the intensive fusion protein.

Description

technical field [0001] The invention belongs to the technical field of medicinal protein. Specifically, the present invention relates to an anti-tumor fusion protein, its coding gene, a drug with the fusion protein as an active ingredient and its pharmaceutical application. Background technique [0002] TRAIL (TNF-related apoptosis-inducing ligand, tumor necrosis factor-related apoptosis-inducing ligand) is a cytokine synthesized by the body itself, and its physiological function is mainly to regulate the body's immune response. TRAIL mainly induces apoptosis through the extracellular apoptotic pathway, but also involves the mitochondrial pathway in the later stage. The TRAIL signaling pathway refers to the use of TRAIL as a ligand to initiate apoptosis signals by binding to death receptors, and finally induce cell apoptosis. Studies have shown that TRAIL and agonist antibodies targeting TRAIL receptors can selectively induce tumor cell apoptosis, while normal tissues are ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62A61K38/17A61P35/00
Inventor 朱大强陈淑珍王晓飞商悦李毅李良
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com